The present invention relates to immunization or vaccination against canine herpesvirus. The invention relates in particular to an immunization or vaccination method, to suitable immunogenic compositions and vaccines and to the use thereof in the context of these methods.
Canine herpesvirus (or CHV) was described for the first time in 1965 (L. E. Carmichael, Am. J. Vet. Res., 1965, 26, 803-814). Infection with canine herpesvirus was then identified in many countries (USA, France, United Kingdom, Switzerland, Italy, Japan, Australia, New Zealand etc.). CHV is responsible for reproductive problems in canine breeding and most particularly for mortality in newborn puppies, which may give rise to significant losses in certain breeding kennels. It is also responsible for abortions and still-births. Infection with CHV is moreover suspected of reducing fertility in bitches. The transmission of CHV takes place via the venereal route, the oronasal route or the transplacental route.
CHV belongs to the family of Herpesviridae and to the subfamily of Alphaherpesvirinae. To date, only one type of CHV has been identified. This is an enveloped virus containing a double-stranded DNA molecule. The envelope contains several glycoproteins of viral origin. The glycoproteins gB, gC and gD are responsible for the induction of neutralizing antibodies in mice.
CHV is antigenically similar to feline herpesvirus (J. A. Limcumpao et al., Arch. Virol., 1990, 111, 165-176) but has no significant cross-neutralization (X. Xuan et al., Arch. Virol., 1992, 122, 359-365).
CHV is strongly dependent on its host and multiplies only on cells of canine origin (Pierson et al., recueil de Mxc3xa9decine Vxc3xa9txc3xa9rinaire, March/April 1998, 174 No. 3/4, 87-94).
Newborn puppies are highly sensitive to infection with CHV during the first 3 weeks of life. The contamination occurs during whelping or the first few days of life. Consequently, it is not possible to vaccinate the puppies. The only means of protection available are hygiene measures such as heating the puppies with an infrared lamp and serotherapy. However, CHV is relatively non-immunogenic and it is difficult to prepare a good serum (L. B. Carmichael, J. Am. Vet. Med. Assoc., 1970, 156, 1714-1721).
Under natural conditions, puppies can be protected with the CHV-neutralizing antibodies present in the colostrum of seropositive bitches: in the absence of these antibodies, the puppies are highly sensitive to infection with CHV (D. L. Huxsoll and I. E. Hemelt, J. Am. Vet. Med. Assoc., 1970, 156, 1706-1713). The at-risk animals are puppies born from uninfected bitches in contact with excreting animals, and puppies born from latent but seronegative or weakly seropositive infected bitches. The CHV-neutralizing antibodies do not persist for a long time, and an animal can be infected and seronegative.
In puppies less than 3 weeks old, CHV causes a neonatal hepesvirosis which is often fatal. The incubation is rapid and the puppies die within a few days, usually with no symptoms other than sudden hypothermia and devastating nervous disorders, in the vast majority of cases before the 5-day-old stage. In the less acute forms, the hypothermia is followed by symptoms such as anorexia, depression, bradycardia, hypoglycemia, subcutaneous edema, erythema and ventral papules, soft yellowish-gray stools, abdominal pains, vomiting, incessant whining, pedaling and death within 24 to 72 hours in opisthotonus. The necropsic examination may reveal an enlarged spleen, serous effusion in the pleural and peritoneal cavities and visceral petechia (Pierson et al., recueil de Mxc3xa9decine Vxc3xa9txc3xa9rinaire, March/April 1998, 174 No. 3/4, 87-94). This article recalls that no vaccine against canine herpesvirosis is currently marketed and suggests that it would be useful to be able to vaccinate latent infected breeders in order to attenuate the re-excretion of the wild-type virus.
On the other hand, in her article (Rec. Med. Vet., 1982, 158, 669-676), F. Delisle proposes vaccinating bitches before gestation with an inactivated vaccine or injecting the puppies with a serum at whelping. She recalls, however, that no commercial vaccine is available. See also L. E. Carmichael, J. Am. Vet. Med. Assoc., 1970, 156, 1714-1721. In his article (xe2x80x9cCanine Herpesvirusxe2x80x9d in Virus Infections of Vertebrates, vol. 1, M. C. Horzinek, Ed. M. J. Appel, Elsevier Science Publisher, 1987, 5-15), A. Appel proposes vaccinating bitches before or at the very start of gestation with an inactivated vaccine.
H. Poulet and P. Dubourget (Point Vxc3xa9t., 1993, 25, 69-75) suggested in 1992, in general terms, the vaccination of the mother with a booster vaccination at the end of gestation. The authors also recall that tests using attenuated variants were carried out (L. E. Carmichael et al., Infection and Immunity, 1978, 20(1), 108-114; U.S. Pat. No. 4,213,965; attenuated live vaccine, composed of a heat-sensitive variant of the CHV virus, referred to as being xe2x80x9cof small plaquexe2x80x9d), but these tests did not yield any convincing results and did not lead to the marketing of vaccines.
In his article (Vet. Med., April 1991, 4, 394-403), J. O. Anvik points out that the production of neutralizing antibodies in the mother and consequently the protection of the litter is unpredictable on account of the low immunogenicity of the CHV virus.
Faced with the difficulty of vaccination, Pierson et al. (recueil de Mxc3xa9decine Vxc3xa9txc3xa9rinaire, March/April 1998, 174 No.3/4, 87-94) even went as far as to propose vaccinating the mother with an inactivated vaccine against felid herpesvirus (FHV) or administering anti-FHV antibodies to puppies at whelping.
To date, none of the vaccination hypotheses has been confirmed, no vaccine is commercially available and no publication has presented convincing vaccination results announcing the impending marketing of such a vaccine.
In addition, in gestating bitches, infection with CHV can lead to embyronic resorption, fetal mummification, abortion or premature whelping, still-birth or neonatal mortality. Placental lesions may appear.
Although vaccines against various herpesviruses exist in other animal species, including cats, it has never been possible to propose effective vaccination against canine herpesvirosis.
The object of the present invention is to develop a method for vaccination against canine herpesvirosis which allows puppies to be protected.
Another object of the invention is to produce efficient and pharmaceutically acceptable vaccines which can be used in the context of this vaccination method.
The Applicant has found, surprisingly, that it is possible to vaccinate gestating bitches and newborn puppies by administering a vaccine during the gestation period and in particular as close as possible to whelping, so as to have a high level of anti-CHV antibodies in the gestating bitch at the time of whelping, this level providing protection to the puppies at whelping by transmission of the maternal antibodies during suckling. In addition, these antibodies are substantially maintained in the puppies during the first weeks of their life, this being the period during which they are prone to disease (3 to 4 weeks). A titer of neutralizing antibodies in the bitch of greater than or equal to 0.9 log10 is preferred, more particularly a titer greater than or equal to 1.2 log10. The titers indicated are calculated according to the method described in Example 7. Vaccination of the bitch moreover leads to a reduction in the pressure of infection in the puppies. The Applicant has moreover observed, in bitches vaccinated and maintained in a contaminated environment, a higher weight of puppies at whelping and a tendency toward a higher level of gestation. These phenomena can be explained by a reduction or absence of placental lesions in the vaccinated bitches (A. Hashimoto et al., Am. J. Vet. Res., 1979, 40(9), 1236-1240; A. Hashimoto et al., Am. J. Vet. Res., 1982, 43(5), 844-850).
The objects of the invention already mentioned, as well as others, are obtained with the aid of a method for immunizing or vaccinating gestating bitches which comprises the administration of an immunogenic composition or vaccine to a gestating bitch as close as possible to whelping, in particular during the final third of gestation (the average duration of gestation in dogs is about 63 days), notably 1 to 20 days, in particular 1 to 15 days, more particularly 5 to 15 days, preferably 5 to 10 days before the presumed date of whelping, typically about 10 days before, so as to have a high level of anti-CHV antibodies in the gestating bitch at whelping, which induces protection of the puppies by transfer of the maternal antibodies during suckling. These antibodies are substantially maintained during the first weeks of life of the puppies, this being the period during which they are prone to disease (3 to 4 weeks).
By definition, an antigen, in an effective amount in the composition or vaccine according to the invention, makes it possible to induce a high and protective level of antibodies under the conditions of the protocol in the gestating bitch and the newborn puppy. This may be an antigen comprising the inactivated CHV virus, the attenuated CHV virus, one or more subunits of the CHV virus or one or more antigens expressed by recombinants.
Preferably, the immunogenic composition or the vaccine is intended to produce in the gestating bitch, at the time of whelping, a level of anti-CHV antibodies (determined according to example 7) of greater than or equal to about 0.9 log10, preferably greater than or equal to about 1.2 log10.
Especially in the case of an animal which has never been vaccinated against CHV, another (or several other) administration(s) of anti-CHV vaccine is preferably carried out before the administration described above in order to induce the immune response in the animal. The interval between these two administrations must be sufficient to induce a booster effect, which generally requires at least 2 to 3 weeks. The first administration of vaccine is notably done within a period which goes from oestrus and at least 2 or 3 weeks before the above described administration, in particular within the period which goes from oestrus to the first third of the gestation, including the first third of gestation, which period includes administration before insemination. Preferably this administration is done about 7 to 10 days before the insemination. According to a preferred embodiment, this vaccination scheme is applied for each whelping occurrence.
If the animal has already been vaccinated against CHV, a single administration close to whelping may suffice.
The administration, or at least one of the additional administrations, can also be carried out with a vaccine whose composition is different from that used as close as possible to whelping, the difference possibly lying in the antigen and/or the formulation of the vaccine.
The composition and vaccine can be administered especially via the parenteral route, preferably via the subcutaneous or intramuscular route.
The dose volumes can be, in particular, between 0.2 and 2 ml, preferably about 1 ml.
A subject of the invention is also the use of CHV antigen to prepare an immunogenic composition or a vaccine against canine herpesvirosis, which is intended to be administered according to the protocol described above.
A person skilled in the art has the competence required to accurately define the doses of each composition or vaccine as a function of the type of composition or vaccine.
When several administrations are performed on the animal, the compositions or vaccines used can be of different types, chosen in particular from those mentioned above.
A subject of the invention is also inactivated immunogenic compositions and vaccines or subunit immunogenic compositions and vaccines, which can be used in particular in the context of this method of vaccination.
The use of CHV subunits has only been described in mice (J. A. Limcumpao et al., J. Vet. Med. Sci., 1991, 53(3), 423-432). However, the data obtained in mice cannot be extrapolated to the canine species, as stated in that document. Although the antibody titers obtained with the purified glycoproteins gp145/112, gp80 and gp47 induce a detectable antibody response, it is not possible, however, to determine whether or not this antibody titer may he reflected by a protection since, as mice are not sensitive to the CHV virus, it is not possible to develop a virulent challenge model in this species. Furthermore, no data are available regarding the cellular immunity induced by CHV. Moreover, the relationship between the antibody titer obtained in mice and that obtained in dogs is unknown and these titers may prove to be different. Thus, it has been published that rabbits immunized with type 1 bovine herpes glycoprotein gI have a higher neutralizing titer than rabbits immunized with gIII, whereas in cattle, the glycoprotein gIV induces the highest neutralizing titer and the glycoprotein gI is the least immunogenic. These data show the difficulty of transposing results from laboratory animals to the target species.
In accordance with the invention, the use of an inactivated immunogenic composition and vaccine or of subunits allows the immunization or vaccination of bitches during the gestation phase without any risk to the bitch or its offspring. Vaccination with booster at the end of gestation is the means for assuring an optimum titer of neutralizing antibodies at whelping. This titer conditions the protection obtained by transfer of neutralizing antibodies from the bitch to its puppies at the start of suckling.
The CHV virus is preferably cultured and propagated on primary canine cells or on a cell line, more particularly on canine kidney cells of a line such as MDCK (Madin-Darby Canine Kidney, available from the American Type Culture Collection (ATCC) under the number CCL-34) in particular with a multiplicity of infection (moi) of from 0.1 to 0.001 tissue culture infective doses 50% (TCID50) per cell, preferably 0.01 TCID50/cell. The CHV virus is harvested 3 to 4 days later. A viral titer of about 106 to 108 TCID50/ml and in general from 106.5 to 107.5 TCID50/ml may be obtained.
In order to obtain an inactivated immunogenic composition or vaccine, the CHV virus is preferably inactivated after harvesting and clarification by means of a chemical treatment (e.g. formalin or formaldehyde, xcex2-propiolactone, ethyleneimine, binary ethyleneimine (BEI)) and/or a heat treatment. Preferably, the virus according to the invention is inactivated by the action of ethyleneimine. The viral particles can be concentrated by conventional concentration techniques, in particular by ultrafiltration and/or purified by conventional purification means, in particular techniques of gel-filtration, ultracentrifugation on a sucrose gradient or selective precipitations, in particular in the presence of polyethylene glycol (PEG).
The subunit immunogenic composition or vaccine is preferably an extraction subunit one, namely is prepared from the whole virus or from a main fraction of this virus (for instance after elimination of capsid). In particular, this immunogenic composition or vaccine may comprise the various envelope glycoproteins or a mixture of envelope glycoproteins. The subunit immunogenic compositions and vaccines are preferably produced from an inactivated viral suspension, in particular inactivated as described above. Preferably, in order to obtain a subunit immunogenic composition or vaccine, mainly containing the envelope glycoproteins, the suspension of viral particles, preferably inactivated, obtained above, is subjected to the action of a suitable detergent, in particular a nonionic detergent, preferably an ethoxylated alcohol advantageously with an HLB of between 12 and 15. The viral capsids are then removed, in particular by ultracentrifugation. Other equivalent methods for removing the capsids and collecting the envelope glycoproteins are available to those skilled in the art.
In order to develop an inactivated immunogenic composition or vaccine or a subunit immunogenic composition or vaccine, the antigen is taken up in a veterinarilly acceptable vehicle or excipient and a veterinarilly acceptable adjuvant is preferably added thereto. The amount of antigen is equivalent in particular to a titer before inactivation of about 105 to about 109.5 TCID50, particularly from about 105 to about 109 TCID50, per dose. More particularly, for an inactivated composition or vaccine, the titer before inactivation is of about 105 to about 109 TCID50, particularly from about 106 to about 108 TCID50, per dose. More particularly for a subunit composition or vaccine, the titer before inactivation is of about 106 to about 109.5, particularly from about 107 to about 109, preferably from about 107.5 to about 108.5, per dose. Preferably, for a subunit vaccine, the vaccine has a glycoprotein gB titre as measured in ELISA of about 0.01 xcexcg to about 30 xcexcg, in particular from about 0.1 xcexcg to about 10 xcexcg, preferably from about 0.3 xcexcg to about 3 xcexcg per dose.
The immunogenic compositions and vaccines according to the invention are conserved and stored either in formulated form at 5xc2x0 C., or in lyophilized form, preferably with a stabilizer, in particular SPGA (sucrose, phosphate, glutamate, albumin, EP-B1-0 008 255). These immunogenic compositions and vaccines may be taken up subsequently in solvent (vehicle or excipient and/or adjuvant).
In order to adjuvate the immunogenic compositions and vaccines according to the invention, it is possible to use (1) aluminum hydroxide, (2) an acrylic acid or methacrylic acid polymer, a polymer of maleic anhydride and of alkenyl derivative, (3) an immunostimulating sequence (ISS), in particular an oligodeoxyribonucleotidic sequence bearing one or more non-methylated CpG groups (Klinman D. M. et al., Proc. Natl. Acad. Sci. USA, 1996, 93, 2879-2883 ; WO-A-9816247), or (4) to formulate the immunogenic or vaccine preparation in the form of an oil-in-water emulsion, in particular the SPT emulsion described on page 147 of xe2x80x9cVaccine Design, The Subunit and Adjuvant Approachxe2x80x9d edited by M. Powell and M. Newman, Plenum Press, 1995, and the emulsion MF59 described on page 183 of this same book.
The oil-in-water emulsion can be based in particular on light liquid paraffin oil (European Pharmacopea type); isoprenoid oil such as squalane or squalene; oil resulting from the oligomerization of alkenes, in particular of isobutene or decene; esters of acids or of alcohols containing a linear alkyl group, more particularly plant oils, ethyl oleate, propylene glycol di(caprylate/caprate), glyceryl tri(caprylate/caprate) or propylene glycol dioleate; esters of branched fatty acids or alcohols, in particular isostearic acid esters. The oil is used in combination with emulsifiers to form the emulsion. The emulsifiers are preferably nonionic surfactants, in particular esters of sorbitan, of mannide (e.g. anhydromannitol oleate), of glycerol, of polyglycerol, of propylene glycol and of oleic, isostearic, ricinoleic or hydroxystearic acid, which are optionally ethoxylated, and polyoxypropylene-polyoxyethylene copolymer blocks, in particular the Pluronic(copyright), block copoymers of propyleneoxide and ethylene, oxide products, especially L121.
The acrylic acid or methacrylic acid polymers are crosslinked in particular with polyalkenyl ethers of sugars or of polyalcohols. These compounds are known under the term xe2x80x9ccarbomerxe2x80x9d (Pharmeuropa vol. 8, No. 2, June 1996). A person skilled in the art may also refer to U.S. Pat. No. 2,909,462 (incorporated by reference) describing such acrylic polymers crosslinked with a polyhydroxylated compound containing at least 3 hydroxyl groups, preferably not more than 8, the hydrogen atoms of at least 3 hydroxyls being replaced with unsaturated aliphatic radicals containing at least 2 carbon atoms. The preferred radicals are those containing from 2 to 4 carbon atoms, e.g. vinyls, allyls and other ethylenically unsaturated groups. The unsaturated radicals can themselves contain other substituents, such as methyl. The products sold under the name Carbopol(copyright), polymers of acrylic acid crosslinked with allylsucrose or allylpentaerythritol, (BF Goodrich, Ohio, USA) are particularly suitable. They are crosslinked with an allyl sucrose or with allylpentaerythritol. Among these, mention may be made of the products Carbopol(copyright) 974P, 934P and 971P.
Among the copolymers of maleic anhydride and of an alkenyl derivative, those which are preferred are the EMA(copyright) products, copolymers of maleic annhydride and of ethylene, (Monsanto) which are copolymers of maleic anhydride and of ethylene, which may be linear or crosslinked, for example crosslinked with divinyl ether. Reference may be made to J. Fields et al., Nature, 186, 778-780, Jun. 4, 1960 (incorporated by reference). As regards their structure, the acrylic acid or methacrylic acid polymers and the EMA(copyright) products are preferably formed from units based on the following formula: 
in which:
R1 and R2, which may be identical or different, represent H or CH3 
x=0 or 1, preferably x=1
y=1 or 2, with x+y=2.
For the EMA(copyright) products, x=0 and y=2. For the carbomers, x=y=1.
The polymer concentration in the final vaccine composition will be from 0.01% to 1.5% W/V, more particularly from 0.05 to 1% W/V, preferably from 0.1 to 0.4% W/V.
One form of immunogenic composition or vaccine which is particularly preferred comprises an inactivated immunogenic composition or vaccine or a subunit immunogenic composition or vaccine formulated in the form of an oil-in-water emulsion comprising anhydromannitol oleate, ethoxylated oleic acid and light liquid paraffin oil. In particular, a method for immunizing gestating bitches according to the invention comprises the administration of such an immunogenic composition or vaccine. Two injections are given, preferably subcutaneously, in particular in accordance with the above-described scheme, for example with the first injection during the first third of the gestation, preferably about 10 days after the date of insemination and in particular the second injection during the final third, preferably about 10 days before the presumed date of whelping.
A subject of the invention is also the process for preparing the inactivated immunogenic compositions and vaccines or the subunit immunogenic compositions and vaccines described.
A subject of the invention is also multivalent immunogenic compositions and vaccines comprising the inactivated canine herpesvirus valency or the canine herpesvirus subunit valency, and at least one valency for at least one other canine pathogen, in a veterinarially acceptable vehicle or excipient and preferably with an adjuvant, especially one of those described above.
Said canine pathogens are chosen in particular from the group comprising canine distemper virus, canine parvovirus and canine adenovirus, but other valencies may also be combined.
The inactivated CHV immunogenic compositions and vaccines or subunit immunogenic compositions and vaccines according to the invention can be administered simultaneously in the same preparation or successively in different preparations with the other canine valency (valencies) which may be in the form of attenuated live, inactivated, subunit, recombinant, or polynucleotide immunogenic compositions and vaccines.
A subject of the invention is thus also a multivalent vaccine kit or pack comprising, separately packaged, a CHV valency according to the invention in a veterinarially acceptable vehicle or excipient, and preferably with an adjuvant, in particular those described above, and at least one valency of at least one other canine pathogen. The CHV valency according to the invention can serve as a solvent for the other canine valency, in particular for an attenuated, recombinant or polynucleotide valency in lyophilized form.
The invention will now be described in greater detail with the aid of embodiments given as non-limiting examples.